Tissue Engineering in Congenital Craniofacial Defects

描述（由申请人提供）：本研究的长期目标是发展组织工程策略，以了解和治疗颅面异常。这个建议既是假设也是设计驱动的。我们将研究小鼠颅面畸形Apert综合征模型细胞的组织发育。我们假设，与在单层或三维水凝胶支架中生长的正常细胞相比，来自co-Pi开发的FGFR2敲入Apert小鼠模型的突变细胞将以明显不同的方式发育组织并对生长因子做出反应。此外，我们假设当正常细胞和突变细胞在多层水凝胶支架中共培养时，增殖、组织发育和生长因子反应将发生变化。该提案的设计驱动部分将开发一种临床实用的策略，用于修复颅面缺陷，在医源性缺陷和疾病小鼠模型中，使用实验室开发的细胞封装技术。为了验证这些假设并设计修复系统，将研究以下具体目标：研究FGFR2敲入小鼠Apert综合征模型分离细胞的细胞行为和组织发育。具体来说，这个目标将解决1.)与来自正常兄弟姐妹和永生化克隆小鼠间充质干细胞系的细胞相比，来自Apert综合征的Fgfr2+/S252W小鼠模型的细胞的软骨形成和成骨。来自a.)长骨和b.)头盖骨的细胞将在体外的2D单层和3D水凝胶支架中进行评估。具体目标2。研究突变细胞和正常细胞在生长因子和共培养作用下的细胞行为和组织发育。具体来说，这一目标将解决1.)在2D和3D水凝胶培养中突变和正常细胞对肌肉骨骼相关生长因子的反应，以及2.)应用一种新的3D双层水凝胶系统共培养正常和突变细胞，以确定共培养对正常和突变细胞的细胞行为和组织发育的影响。具体目标3。测定颅面模型中突变细胞和正常细胞的体内功能。具体来说，1.医源性小鼠颅骨缺陷和2. Apert综合征先天性缺陷的组织发育将使用包埋在水凝胶中的细胞进行评估。除了克隆衍生的小鼠间充质干细胞外，还将使用正常和突变小鼠细胞系。组织发育将通过放射学和组织学分析进行评估。

DESCRIPTION (provided by applicant): The long term objective of this research is to develop tissue engineering strategies for the understanding and treatment of craniofacial abnormalities. This proposal is both hypothesis and design driven. We will investigate tissue development of cells from mouse models of the craniofacial abnormality Apert syndrome. We hypothesize that mutant cells from the FGFR2 knock-in Apert mouse model developed by the co-Pi will develop tissue and respond to growth factors in a significantly different manner compared to normal cells when grown in monolayer or in three dimensional (3D) hydrogel scaffolds. Furthermore, we hypothesize that when the normal and mutant cells are cocultured in multilayered hydrogel scaffolds, proliferation, tissue development, and growth factor response will change. The design driven portion of the proposal will develop a clinically practical strategy for repair of craniofacial defects, in iatrogenic defects and in the disease mouse model, using cell encapsulation technologies developed by the lab. To test these hypotheses and design the repair system the following specific aims will be investigated: Specific aim 1. Investigate cell behavior and tissue development of cells isolated from FGFR2 knock-in mouse model of Apert Syndrome. Specifically this aim will address 1.) chondrogenesis and osteogenesis of cells from a Fgfr2+/S252W mouse model of Apert Syndrome, compared to cells from normal siblings and an immortalized, clonal mouse mesenchymal stem cell line. Cells from a.) the long bone and b.) the cranium will be evaluated in 2D monolayer and 3D hydrogel scaffolds in vitro. Specific aim 2. Investigate cell behavior and tissue development of mutant and normal cells in response to growth factors and co-culture. Specifically this aim will address 1.) response of mutant and normal cells to musculoskeletal-related growth factors in 2D and 3D hydrogel culture, and 2.) apply a novel 3D bilayered hydrogel system to coculture normal and mutant cells to determine the effects of coculture on cell behavior and tissue development of normal and mutant cells. Specific aim 3. Determine in vivo function of mutant and normal cells in craniofacial models. Specifically, tissue development in 1.) iatrogenic mouse cranial defects and 2.) congenital defects in Apert Syndrome will be evaluated using cells embedded in hydrogels. Normal and mutant mouse cell lines will be used in addition to a clonally-derived mouse mesenchymal stem cell line. Tissue development will be assessed by radiographic and histological analyses.